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DR SIMON CLULOW (Orcid ID : 0000-0002-5700-6345)
Article type
: Research Article
Handling Editor: Hamish McCallum
Elevated salinity blocks pathogen transmission and improves host survival from the global
amphibian chytrid pandemic: implications for translocations
Simon Clulow1*, John Gould1, Hugh James1, Michelle Stockwell1, John Clulow1 & Michael
Mahony1
1
School of Environmental and Life Sciences, University of Newcastle, Callaghan NSW 2308,
Australia
*
Corresponding author email: simon.clulow@newcastle.edu.au
Abstract
1. Emerging infectious diseases are one of the greatest threats to global biodiversity.
Chytridiomycosis in amphibians is perhaps the most extreme example of this phenomenon
known to science. Translocations are increasingly used to fight disease-induced extinctions.
However, many programs fail because disease is still present or subsequently establishes in
the translocation environment. There is a need for studies in real-world scenarios to test
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doi: 10.1111/1365-2664.13030
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whether environmental manipulation could improve survival in populations by generating
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unfavourable environmental conditions for pathogens. Reintroductions of amphibians
impacted by chytridiomycosis into environments where the disease persists provide a
scenario where this paradigm can be tested.
2. We tested the hypothesis that manipulating environmental salinity in outdoor mesocosms
under near identical environmental conditions, present in a nearby translocation program for
an endangered amphibian, would improve survival and determine the mechanisms involved.
160 infected and 288 uninfected, captive-bred, juvenile frogs were released into 16 outdoor
mesocosms in which salinity was controlled (high or low salinity treatment). The experiment
was run for 25 weeks from the mid-austral winter to the mid-austral summer of 2013 in a
temperate coastal environment, Australia.
3. Increasing salinity from ca. 0.5 ppt to 3.5 - 4.5 ppt reduced pathogen transmission between
infected and uninfected animals, resulting in significantly reduced mortality in elevated salt
mesocosms (0.13, high salt versus 0.23, low salt survival at 23 weeks). Increasing water
temperature associated with season (from mean 13oC to 25oC) eventually cleared all
surviving animals of the pathogen.
4. Synthesis and applications. We identified a mechanism by which environmental salinity
can protect amphibian hosts from chytridomycosis by reducing disease transmission rates.
We conclude that manipulating environmental salinity in landscapes where chytrid-affected
amphibians are currently translocated could improve the probability of population persistence
for hundreds of species. More broadly, we provide support for the paradigm that
environmental manipulation can be used to mitigate the impact of emerging infectious
diseases.
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Keywords: amphibian, Batrachochytrium dendrobatidis, chytrid, chytridiomycosis, disease,
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environmental mitigation, refugia, reintroduction, translocation, transmission
Introduction
Mounting evidence indicates that novel infectious diseases can alter the demographic
trajectories of naïve populations, leading them to become unstable and at risk of extinction
(Smith, Sax & Lafferty 2006). Chytridiomycosis in amphibians (Berger et al. 1998; Olson et
al. 2013) is perhaps the most extreme example of this phenomenon known to science, given
the massive decline and extinction of hundreds of species due to this single pathogen (Stuart
et al. 2004; Lips et al. 2006; Skerratt et al. 2007; Vredenburg et al. 2010; Alroy 2015; Bower
et al. 2017). Sometimes, the impacts of diseases such as chytridiomycosis are not uniform
across the ranges of species, and species persist in remnants of former distributions, which
effectively become refugia (Briggs et al. 2005; Rowley & Alford 2007; Rödder, Veith &
Lötters 2008; Puschendorf et al. 2009; Briggs, Knapp & Vredenburg 2010; Tobler & Schmidt
2010; Puschendorf et al. 2011; Scheele et al. 2014a; Bower et al. 2017). Understanding the
drivers of disease dynamics in remnant populations destabilised through the co-existence of
pathogen and host is thus an important area of investigation in conservation biology and
disease ecology because it offers the potential for improved outcomes for species impacted
by emerging diseases.
The ongoing impact of pathogens on host post-emergence population dynamics is determined
by the transmission rate (Begon et al. 2002; Woolhouse, Haydon & Antia 2005) and levels of
pathogen load, morbidity and mortality expressed in infected individuals (reflected in
survivorship). In theory, it may be possible to mitigate the impacts of a pathogen on a host
population by altering components of the environment of the host if they reduce the rates of
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transmission, morbidity and mortality. There is evidence that environmental factors mitigate
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the biological activity of Batrachochytrium dendrobatidis (Bd), the aetiological agent in
chytridiomycosis, and as a result the disease dynamics in host populations. The viability and
disease impact of the aquatically transmitted Bd may be inhibited and limited by elevated
temperature (Johnson et al. 2003; Piotrowski, Annis & Longcore 2004; Forrest & Schlaepfer
2011; Savage, Sredl & Zamudio 2011; Stevenson et al. 2013) and salinity (Stockwell, Clulow
& Mahony 2012; Heard et al. 2014; Stockwell, Clulow & Mahony 2015; Stockwell et al.
2015). As well, there is evidence that post-metamorphic stages of some susceptible host
species may be cleared of the disease in wild populations (Briggs et al. 2005; Kriger & Hero
2006; Murray et al. 2009; Briggs, Knapp & Vredenburg 2010; Brannelly et al. 2015). This
suggests that manipulation of environmental temperature and salinity may alter vital rates
under some circumstances in a manner that improves the demographic status of some
impacted amphibian populations and species.
A popular conservation strategy globally for declining wildlife is to reintroduce or translocate
individuals of affected species to new or formerly-occupied habitats (Germano et al. 2015).
Often, as is especially the case with mitigation-driven translocations, such habitats are
purpose-built or modified specifically for translocation purposes (Germano et al. 2015),
providing a unique opportunity to incorporate design features that could mitigate threats such
as disease. Considering that translocations are a popular conservation strategy for endangered
amphibians (Germano & Bishop 2009), and that many such programs fail due to the presence
of disease (Stockwell et al. 2008), understanding whether, and by what mechanisms,
environmental manipulations could mitigate such impacts in real-world scenarios is an
important conservation goal.
The green and golden bell frog (Litoria aurea) is one species in which there has been a major
spatial decline in its range due to chytridiomycosis, with co-existence of pathogen and host in
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all but one of its persisting populations (Mahony 1999; Stockwell et al. 2008; Mahony et al.
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2013). In the case of L. aurea, chytridiomycosis appears to be the proximate cause of this
decline, and ongoing population instability (Mahony et al. 2013). This species is no longer
encountered over more than 90% of its former range, but persists in what appears to be
demographically unstable remnant populations close to the coast in New South Wales and
eastern Victoria, Australia (Mahony et al. 2013). Over time, some of those persisting
populations are going extinct, while few, if any remaining ones can be regarded as secure
(White & Pyke 2008; Mahony et al. 2013). Co-existence of pathogen and host in remnant
populations arguably leads to ongoing demographic instability in those populations (Bower et
al. 2013), and understanding this is complicated by a temporal (seasonal) component to
disease expression and impact (Stockwell et al. 2008), as well as spatial heterogeneity of
environmental variables such as salinity that may affect survival and demographic parameters
(Stockwell, Clulow & Mahony 2015). Considering that this species has been subject to
numerous translocation and reintroduction attempts (McFadden et al. 2008; Germano et al.
2015; James et al. 2015), many of which have failed due to the ongoing presence of Bd in the
wild (Stockwell et al. 2008), it is an ideal species to explore the concept of environmental
manipulation to mitigate the threat of disease.
We carried out an experiment using open mesocosms located within 5 km of a persisting, but
unstable, population of L. aurea (from which captive bred animals were derived to perform
the current study) to test whether altering environmental salinity could influence disease
dynamics (pathogen transmission and host survivorship) and improve translocation success.
The source population is close to the mouth of the Hunter River, NSW Australia (32° 51′ 49
S, 151° 44′ 29 E) and thus subject to maritime influences on water body salinities and
climate, both of which may be factors in the persistence of the species (Lane, Hamer &
Mahony 2007; Hamer, Lane & Mahony 2008; Stockwell, Clulow & Mahony 2015).
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Specifically, we aimed to determine whether salinity reduced Bd transmission or provided a
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curative effect (or both) in real-world environments and thus increased survival, which might
explain persistence of L. aurea in coastal environments. The experiment was conducted
commencing in the austral winter, and concluding in the austral summer to expose the frogs
to the maximal seasonal thermal challenge that this species faces in the geographical location
of the source population. All of our environmental manipulations were carried out in such a
way that might realistically be incorporated into translocation programs for threatened
amphibians in the future.
Materials and Methods
Source of animals and captive husbandry
Juvenile green and golden bell frogs Litoria aurea were grown from tadpoles bred at the
University of Newcastle (animal ethics approval A-2013-302; NSW NPWS licence
SL100190). Tadpoles were raised in outdoor tubs (200cm x 1100cm x 60cm) and fed a
mixture of ground and whole trout pellets (Ridley Aqua-Feed, Ridley AgriProducts Pty Ltd,
Narangba, Australia) ad libitum. Metamorphosing individuals (Gosner stage 42) were moved
into the laboratory where they were housed in small plastic aquaria (30cm x 20cm x 15cm)
with gravel and aged tap water to a depth of 5cm. Once each metamorph had undergone full
tail regression and reached a minimum SVL of 25mm, a small passive integrated transponder
(Hongteng HT-157 PIT tag, Hongteng, Gangzhou) was positioned under the skin of the right
lateral surface for identification. Juvenile frogs were fed live crickets (Acheta domesticus,
Pisces Enterprises, Brisbane) ad libitum and maintained at room temperature on a 12hr day
(5% UV lamp)/night cycle.
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Cultivation of Chytrid fungus (Batrachochytrium dendrobatidis)
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Batrachochytrium dendrobatidis (strain: Gibbo River-Llesueuri-00-LB-1) was derived from
pre-existing stock held at the University of Newcastle, NSW. In vitro cultivation was carried
out by seeding TGhL agar plates (16g tryptone, 4g gelatine hydrolysate, 2g lactose, 10g
bacteriological agar, and 200mg penicillin-G in 1000ml distilled water) with 2ml of a oneweek-old actively growing liquid chytrid broth. Plates were incubated at 17-19°C and
periodically checked for growth using an inverted light microscope until colonies of
zoosporangia and free-swimming zoospores could be detected. A zoospore suspension was
then prepared by flooding inculcated TGhL agar plates with 1.5ml of liquid media (TGhL).
These plates were left to stand for 5mins, before the supernatant was collected.
Chytrid infection
Two hundred and fifty juvenile frogs were moved from the laboratory aquaria in which they
were held after metamorphosis to new containers filled with gravel and tap water for
infection with Bd. Approximately 200µl of Bd zoospore suspension was pipetted into the
water of each container. An additional 250 frogs that were to remain uninfected prior to
introduction to outdoor mesocosms were placed in identical containers to which an equivalent
volume of a sterile agar broth solution without Bd zoospores was added. Water changes
ceased for a period of 5 days to allow animals contact with the inoculated water to acquire
infection. A subset of frogs from each of the ‘infected’ and ‘uninfected’ containers were
swabbed to test for the presence of Bd seven days after exposure to determine if frogs had
become infected. This time interval is sufficient to allow the Bd to undergo a complete life
cycle of reproduction in infected animals (Longcore, Pessier & Nichols 1999; Piotrowski,
Annis & Longcore 2004). Upon analysis of the swabbing data (see below for details), it was
found that no individual tested positive for infection. Animals were, therefore, exposed to an
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additional 200µl of a zoospore suspension two weeks later and re-swabbed. After this
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exposure period, it was found that frogs from two treatment containers tested positive for Bd.
Individuals from these containers were distributed evenly amongst the other ‘infected’
containers to allow for the natural spread of the pathogen, which was shown to occur for all
animals in ‘infected’ tanks via swab testing approximately two weeks later. ‘Uninfected’
containers remained free of Bd. The experiment commenced shortly after confirming the
infection status of all animals (within one week).
Outdoor mesocosm configuration
Sixteen cylindrical polyethylene mesocosms (Duraplus Aquapoly Aquaculture tubs: 3.5 m
diameter, 1 m height, 10,000 L volume) were filled over half their bottom surface area with
gravel to a depth of 30 cm; with the remaining half of the bottom surface filled with aged rain
water to a maximum depth of 30 cm (Fig. 1). Habitat was provided in the form of brick piles
and plastic plants which were placed in three rows - on the dry gravel, along the shoreline and
within the water body. Bricks were placed individually (flat on the ground and perpendicular
to the water) and in groups consisting of five bricks stacked in a lattice formation (three
bricks laid 1 cm apart, parallel and perpendicular to the water, with two bricks laid on top at
right angles to the bricks below; Fig. 1). These brick piles have been shown to be a preferred
refuge of juvenile L. aurea in many outdoor field trials that we have conducted (data not
presented). A small drainage hole was drilled through the wall of the tub to enable excess
water to drain out of the mesocosm during periods of heavy rainfall, and to allow
maintenance of a constant water level in each mesocom. Each tub was covered with heavy
netting to form an enclosed system, with the connection between the netting and rim of the
tub bordered with an additional 10 cm wide strip of fine mosquito netting to prevent frog
escapes.
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Experimental design
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Each salinity treatment (high or low) was replicated across eight mesocosms, with treatments
assigned randomly using a random numbers generator in Excel. Natural sea salt (Cheetham
Salt Limited, Sunray Swimming Pool Salt) was added to the ‘high salinity’ tubs to achieve a
concentration of approximately 4ppt (target range: 3.5-4.5 ppt). Non-salted water bodies
(‘low salinity’ treatments) were found to fluctuate between 0-1ppt. These ranges of salt levels
in waterbodies occupied by L. aurea are observed regularly in the field in natural L. aurea
ponds and are within the known physiological limits of the species (Mahony et al. 2013;
Stockwell, Clulow & Mahony 2015). Salinity values were recorded periodically across the
study period using a water quality meter (YSI professional plus water meter, Xylem, USA) to
measure salt levels, and additional water or salt was added where necessary to maintain the
targeted salt concentrations. Water temperatures were monitored every ten minutes during the
study period using temperature data loggers (iButton®) wrapped in a single layer of paraffin
film and positioned along the central line of tub, 10cm away from the edge (deepest point).
Originally, a two-way factorial experiment was planned between water temperature
(high/low) and salinity (high/low) as the main effects. However, attempts to heat the water in
mesocosms passively using solar powered pumps to circulate water through black polypipe
exposed to the sun (considered a potential option in the field for created habitats for
reintroduction programs) and by using aquarium heaters powered by a diesel generator failed
to heat the water bodies in ‘high temperature’ treatments by more than 0.5° C mean daily
difference compared to ‘low temperature’ treatments during the study period, despite a
difference of 3-4° C being achieved during preliminary trials. Thus, the experiment
effectively became a salinity only experiment.
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Animal release into mesocosms
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Prior to release, the PIT number, initial weight (g), snout-vent length, head-width and right
tibia length (recorded to the nearest mm using dial callipers) of infected and non-infected
frogs were recorded. Each individual was swabbed to test for Bd infection immediately prior
to release by swabbing the hands, feet, thighs and lateral sides with a sterile swab (Tubed
Sterile Dryswab™ Tip MW100) in a standardised manner, involving 4 strokes posterior to
anterior and back (8 strokes total) of each of the lateral sides and inner and outer thighs, and 4
strokes (applied in a twirling motion) of the hands and feet. Eighteen non-infected frogs and
10 infected frogs were released into each of the experimental mesocosms on the 12th of July,
2013 (the middle of the austral winter). Individuals were distributed randomly among the 16
mesocosms to avoid bias in allocating to treatments arising from variations in size, sex and
age at the time of release. All animals were released separately into the water so that they
were not forced to come into contact with other frogs at the time of release. The experiment
was terminated on the 24th of December (mid-summer), 2013. The start and end dates of the
experiment were chosen due to observations in the wild that Bd infection prevalence and
loads (and resulting effects of chytridiomycosis) are most intense in the winter months and
largely benign in the summer months (believed to be linked to temperature; (Stockwell et al.
2008; Mahony et al. 2013). Thus, disease intervention is most likely to be optimal during the
winter months.
Mesocosm surveys
A census of the experimental mesocosms was carried out at 1, 2, 5, 8, 13, 18 and 23 weeks
post-release. During each census, each frog was caught by hand in an individual plastic bag
to prevent cross-contamination of frogs during handling before being identified by PIT
number, weighed, measured and swabbed as per the standardised method above. Frogs were
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then released back onto the gravel surface of their respective tubs, making sure that no
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individual made contact with another to prevent cross-infection during the release. Mesocosm
tubs were checked daily for dead or moribund frogs. These were collected, identified by PIT
tag number, swabbed and, if alive but moribund, euthanized. The diets of animals in
mesocosms were supplemented with live crickets ad libitum throughout the experiment.
Detection of Chytrid infection by real-time qPCR analysis of swabs
Nucleic acids were extracted from swabs and Bd DNA quantified using a qPCR Taqman
assay (Boyle et al., 2004). Each swab sample was analysed in triplicate using a Rotor Gene
6000 real time DNA amplification system (Corbett Life Science, Sydney, Australia)
following a 1/10 dilution of the original DNA extract. After amplification, the number of B.
dendrobatidis genomic equivalents (GE) detected at a standardised cycle threshold was
calculated across all three replicates as a geometric mean in samples considered positive.
Samples were considered positive for B. dendrobatidis when amplification occurred in at
least two of the three replicates. Non-positive (zero) values were included in the calculation
of the geometric mean as these were assumed to be the result of a low quantity of DNA
present within the sample. For all samples, the mean GE value was multiplied by 10 to
account for the dilution step carried out during the extraction process.
Samples were considered negative for the presence of Bd if the process of amplification did
not occur in two or more of the three replicates from each sample, providing that the PCR
reaction was not inhibited. In order to detect such inhibition, an internal positive control was
tested in a single replicate of each sample, along with one replicate of the negative B.
dendrobatidis template control. Following qPCR, the data for the cycle number in which the
sample crossed a threshold set halfway up the amplification curve was noted. Samples were
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considered inhibited if the amplification curves for positive controls crossed the threshold
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point more than five cycles after that of the negative control.
Data analyses
Univariate survival analysis was initially carried out using Kaplan-Meier plots in JMP
(version 11), testing separately for the effects of salt for all frogs released, as well as frogs
released initially as infected and uninfected alone. Log-Rank and Wilcoxon tests were used to
determine the significance of differences between treatments in the survival curves over time.
The proportion of individuals infected with chytrid over time was analysed using a mixed
effects logistic regression model in SAS version 9.4 (glimmix procedure), with time and salt
treatment as fixed effects and a random effect for mesocosm. The repeated measures per
individual were modelled using a residual covariance matrix with autoregressive order one
covariance structure. The Kenwood-Roger adjustment was applied to correct for downward
bias in the variance-covariance matrix.
Log of infection load was used to correct for skewness in infection load data. Infection load
within the infected proportion of the population was analysed over time using linear mixed
effects models (LMM) in SAS (PROC MIXED procedure), with salt and time and all twoway interactions as fixed effects. Mesocosms were added as a random effect and repeated
measures over time were modelled with a residual covariance matrix with compound
symmetry covariance structure.
Effects of the salt treatments on L. aurea growth rates were examined using linear mixed
effects models (LMM) in SAS, with a full factorial model run for the effects of salt and time
on tibia length, head width and snout-vent length.
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Differences in salt levels between ‘high’ and ‘low’ treatments were investigated using t-tests
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in Excel.
Results
Fluctuations in salt concentrations and seasonal water temperature
Mean salt concentrations in ‘high salt’ treatment mesocosms were consistently higher than
those in ‘low salt’ mesocosms (p < 0.001; Fig. 2). Mean high salt treatment concentrations
generally fluctuated between 3.5 – 4.5 ppt, except for a drop to around 2 ppt in week 19
following a sustained heavy rainfall event (Fig. 2). Low salt treatment means generally
fluctuated around 0.5 ppt and rarely exceeded 1 ppt (Fig. 2). Mean weekly water temperature
increased from approximately 13°C to approximately 25°C across the study period, which ran
from mid-winter to mid-summer (Fig. 3).
Effect of salt and waterbody temperature on morphometrics
Neither salt nor water temperature effects resulted in any significant differences between
treatments for any of the morphometrics (tibia length, head width or snout-vent length) over
time.
Effect of salt concentration on survival
Litoria aurea survival was higher in mesocosms containing high salt concentrations after 23
weeks than in those containing low salt concentrations (Fig. 4a). Survival was 0.23 in the
high salt treatment at 23 weeks compared to 0.13 in the low salt treatment. The Wilcoxin test
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for determining the significance of differences in the survival curves suggested that these
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survival curves were not significantly different between salt treatments (2 = 1.04, d.f. = 1, P
= 0.31), while the Log-Rank test suggested that they were significantly different (2 = 4.66,
d.f. = 1, P = 0.03). The different outcomes of the two tests are likely due to the breaking of
the proportional hazard assumption implicit in these tests (i.e. through the crossing over of
the survival curves early on in the experimental period) which are handled differently by each
test. The Wilcoxin test places more emphasis on the early part of the survival curves while
the Log-Rank test places emphasis more evenly across the experimental period. Taking both
tests together, it can be suggested that there was no significant difference in the early period
of the survival curves, but that the curves deviated significantly in the latter period of the
experiment.
Almost all of the increased survival in the high salt treatments was attributable to the higher
survival rates of animals that were initially released into the mesocosms without Bd infection
(Fig. 4b). These animals experienced survival of 0.34 in high salt treatments at 23 weeks
compared to 0.20 in low salt treatments. For the animals released initially without Bd, this
difference was found to be significant with both the Log-Rank test (2 = 6.36, d.f. = 1, P =
0.01) and Wilcoxin test (2 = 4.37, d.f. = 1, P = 0.04). Animals initially released with Bd
infection had similarly low survival irrespective of salt treatment, with 0.04 survival in high
salt compared to 0.01 in low salt treatments by 23 weeks (Fig. 4c). These survival curves
were not significantly different by either test (Log-Rank 2 = 0.33, d.f. = 1, P = 0.56;
Wilcoxin 2 = 0.003, d.f. = 1, P = 0.96). Collectively, survival was much higher overall in
animals initially released without Bd infection compared to those released with Bd infection
(Figs. 4b & 4c).
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The rate at which survival decreased also changed markedly between the two groups of
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animals. Survival in the initially uninfected animals decreased very slowly in the first 5
weeks, with survival decreasing to 0.77 and 0.72 in low and high salt respectively before
decreasing rapidly in the low salt treatment to 0.34 by week 8 (Fig. 4b). In contrast, the
survival of uninfected animals released into the high salt treatment only declined to 0.59 by
week 8. The survival for the Bd infected animals on the other hand decreased very rapidly,
down to 0.09 and 0.14 in low and high salt respectively in the first 5 weeks alone (Fig. 4c),
and very few initially Bd infected animals survived to week 23 in either treatment.
Clearance of infection from surviving animals over time
There was a strong effect of time on the proportion of individuals in the mesocosm
population that were infected with Bd (F5, 1249 = 53.42, P < 0.001; Fig. 5a). The population
experienced a rapid increase in the prevalence (i.e. proportion of individuals infected) of Bd
infection by week 8 (early September) before a decrease shortly after week 13 (mid-October)
when infected individuals either died or clear themselves of the infection, with no animals
infected by week 23 (late December; Fig. 5a). The clearance of infection from the mesocosm
population was associated with the seasonal increase in mean water temperature across the
experimental period (Fig. 3).
The overall proportion of infected individuals in the mesocosm population over time was
consistently higher in the low salt treatment than the high salt treatment (Fig. 5a), although
this was not found to be statistically significant with α = 0.05 (F1, 44.13 = 2.92, P = 0.09). The
size of this effect as characterised by the odds ratio was 0.78 [95% CI = 0.55 - 1.11],
indicating that the chance of Bd infection in the high salt treatment was 0.78 times, or 22%
lower than that of the low salt treatment.
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There was also a strong effect of time on Bd infection load within the infected mesocosm
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population (F5, 764 = 92.68, P < 0.001; Fig. 5b). This effect closely matched the pattern of
infection prevalence, with infection loads increasing rapidly in infected animals with peaks
between 8 and 13 weeks post-release (Fig. 5b). Declines in infection load occurred after week
8 and were close to zero by week 13, which is the point at which the population began to
clear itself of infection (Fig. 5a). Salt did not have an effect on Bd load over time (F1, 27.9 =
0.03, P = 0.86; Fig. 5c).
Differences in rates of infection state transitions between high and low salt treatments
The patterns of change of infection state within individuals in the population (i.e. from
uninfected to infected or vice versa) differed over time (Table 1). A much higher proportion
of the mesocosm population in both low and high salt treatments changed from uninfected to
infected at each survey period in the first 8 weeks of the experiment (when the population
was nearing its infection prevalence peak), before switching to a state in which a much
greater proportion of the population cleared itself of the infection (changing from infected to
uninfected) after week 8 (Table 1). All of the surviving animals in both salt treatments
changed infection state from infected to uninfected between weeks 13 and 18 (Table 1). In
all, bar one, time period (week 5 to week 8), the proportion of animals changing from
uninfected to infected was lower in the high salt treatment (Table 1).
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Discussion
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This study has shown that it is possible to manipulate salinity levels in a realistic
environmental setting in a way that can lead to significant improvements in host survival for
a threatened frog that coexists with Bd and is impacted by chytridiomycosis. Importantly, for
the first time, we have demonstrated that the probable primary mechanism of a previously
demonstrated beneficial effect of salinity on survival of L. aurea, is reduced disease
transmission rather than increased survival of infected frogs (i.e. there appears to be no
curative effect for already infected individuals in a field setting). This is an important finding
for the management of species in which there has been a shift in demographics with a
reduced proportion of adults in reproductively mature age classes due to chytridiomycosis.
Based on the results of this study, there may be a measurable gain in the population viability
of host species if salinity in water bodies can be increased to a level where the viability of the
pathogen is sufficiently reduced to reduce transmission rates. This could become an
important management strategy for not only managing the threat of affected species that coexist with the disease in existing populations and habitats, but in particular could be
incorporated into created or modified habitats that form the basis of a swathe of translocation
programs around the globe (Germano et al. 2015), provided that the salinity levels are within
the physiological limits of the host species.
The increased survival in the high salt treatment in this study was not trivial; an increase in
survival from 13% to 23 % among juvenile L. aurea through the winter months represents a
relative increase in survival of 77% over that time period compared to the low salt treatment.
Such an increase in survival could translate into large differences in the number of juvenile
frogs that survive the winter period post-metamorphosis, and adults in the population capable
of contributing to recruitment through reproduction if applied to the source population.
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Modelling and field studies by Pickett et al. (2014) on another coastal population of L. aurea
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located approximately 200 km south of the population/outdoor mesocosms in this study
support this view. That modelling indicated that an increase in female survivorship at 2 years
(the age at which females reach sexual maturity) and beyond into older adult age classes
could lead to a significant improvement in the population viability and a reduced probability
of extinction in L. aurea populations.
The salinity range in this study (0 - 4 ppt) was similar to a previous study in ponds of a
nearby population which showed lower Bd loads and increased survival of L. aurea at 2-4 ppt
compared to ponds with salinities at or close to 0 ppt (Stockwell et al. 2015), although the
mechanism by which this occurred (curative effect or reduced disease transmission) was not
determined. However, the timing of the release and establishment of L. aurea progeny into
the ponds of the earlier study was different to the current study. In the earlier study, L. aurea
were released as tadpoles in the middle of summer (also in different years to the current
study), and growth, infection status and survival was monitored over one year from summer
to summer. An advantage of the current study was that juvenile frogs were released in midwinter, at a time when the impact of chytridiomycosis is known to be at its seasonal peak in
L. aurea (Stockwell et al. 2008; Mahony et al. 2013), as in many other susceptible species. In
addition, in the current study, a combination of both Bd-infected and Bd-free animals were
released together to specifically study the effects of saline influences on infection
transmission. The demonstration in this study that transmission is reduced under conditions
of elevated salinity during the season when the transmission and mortality rates are highest
was possible because of the timing and design of the study. Further, commencing this study
in mid-winter and following the survival and infection dynamics until mid-summer showed
that infected L. aurea that survive winter are capable of clearing the disease at a high rate
This article is protected by copyright. All rights reserved.
heading into summer. This reinforces the view supported by some published data on L. aurea
Accepted Article
(Stockwell et al. 2008) that increased mortality in colder months of the year was the driving
force of demographic shifts in the species responsible for its decline across its former range.
It is worth noting that attempts to manipulate water temperature in this study did not produce
a large enough thermal shift to directly confirm this as an experimental treatment, despite
attempts by several methods that might have been able to be applied in a field setting. This is
important because active and passive strategies have been proposed in the literature to raise
air and water temperatures, such as using dark substrates in ponds and removal of aquatic and
overhanging terrestrial vegetation to reduce shading and increase exposure to solar radiance
(Raffel et al. 2010; Becker & Zamudio 2011; Becker et al. 2012; Heard et al. 2014; Scheele
et al. 2014b). Our experience suggests that directly managing temperature in external
waterbodies by passive or active management of temperature will be a challenging
environmental management strategy to implement, even if a potentially important one
(Scheele et al. 2014b). Such an investigation awaits the development of a better system for
heating waterbodies in outdoor environments.
Few studies have dealt with the manipulation of environmental salinity as a management
approach for mitigating Bd load and impact in habitats, although it has been suggested
(Stockwell, Clulow & Mahony 2012; Scheele et al. 2014b; Stockwell, Clulow & Mahony
2015; Stockwell et al. 2015). The susceptibility of Bd to increasing salinity has been
demonstrated in culture through reduced growth rates and motility/viability (Johnson et al.
2003; Stockwell, Clulow & Mahony 2012) and there is evidence in support of benefits of
salinity to frogs and tadpoles in some studies (Stockwell, Clulow & Mahony 2012; Heard et
al. 2014; Stockwell, Clulow & Mahony 2015; Stockwell et al. 2015) in addition to the current
This article is protected by copyright. All rights reserved.
study. It is worth noting that there are probably scenarios where manipulating salinity on a
Accepted Article
scale that would be detectable in systems where salinity is naturally low could be difficult.
Nevertheless, some systems such as coastal estuarine systems that are a mosaic of oligohaline
and fresh water, as is the case in the environment of many persisting L. aurea populations
(Valdez et al. 2015; Klop-Toker et al. 2016), may offer opportunities to pursue this as a
passive strategy e.g. by manipulation of tidal flows and inundation at a landscape level, or
actively by the addition of sea salt to waterbodies.
The findings of this study suggests that where attempts are made to reintroduce or establish
new populations of amphibians to secure species from the pandemic disease Bd, these should
have the best chance of success in environments where salinities can be increased either
passively or manually to reduce infection rates and transmission, and lead to higher pathogen
clearance rates and adult survival. Such effects, mediated through salinity may be employed
to benefit the demographics by shifting the age class structure towards older animals. On the
basis of the results of this study, addition of salt to water bodies (to achieve concentrations of
ca. 2-4 ppt) should be considered for incorporation into the design of constructed
supplementary or modified habitats for the management of amphibians sensitive to
chytridiomycosis, where it is feasible to do so. This study demonstrated a fundamental
principle in relation to management of species whose conservation status is impacted by
emerging disease, but which persist, albeit at a higher risk of extinction. Understanding
mechanisms of disease transmission and dynamics as they play out in realistic environmental
scenarios is a strategy worth pursuing, since such investigations may identify management
strategies that increase resilience of susceptible species at the landscape level.
This article is protected by copyright. All rights reserved.
Acknowledgements
Accepted Article
We thank Tim Callaghan for assistance with carrying out surveys. Port Waratah Coal
Services provided funding for the study.
Data accessibility
Data are available in the Dryad Digital Repository. DOI: 10.5061/dryad.r0904
(Clulow et al. 2017)
Authors’ Contributions
SC, JC, MS and MM conceived the ideas and designed methodology; SC, JG, and HJ carried out the
experiments and collected the data; SC analysed the data and led the writing of the manuscript. All
authors contributed critically to the drafts and gave final approval for publication.
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Tables
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Table 1: Changes in Infection State in surviving frogs within intervals from 0 to 23 weeks in
Low and High Salt treatments. N indicates uninfected frogs (negative for Bd). I indicates
infected (Bd swab positive) frogs. # = number of individuals that changed state in the
indicated interval (numerator = number changing state; denominator = number in starting
state at beginning of respective interval); % = the percentage of individuals in one state (N or
I) that changed to another state (N or I) within the indicated interval. Blank cells indicate no
change of state was possible within particular intervals due to absence of frogs in the starting
state in respective treatment in that interval. Mean water temperature is across all mesocosms
in all treatments for the second week in each interval.
Salt
Time (weeks)
State change
0-1
1-2
2-5
N to I (#)
27/101
50/72
29/31
N to I (% of N)
27%
69%
I to N (#)
9/41
I to N (% of I)
Number Alive
5-8
8 - 13
13 - 18
18 - 23
2/4
0/17
0/24
94%
50%
0%
0%
9/51
4/76
7/75
19/28
7/7
22%
18%
5%
9%
68%
100%
142
123
107
79
28
24
24
26/118 53/101
44/51
4/7
0/8
0/62
0/57
0%
Low
N to I (#)
N to I (% of N)
22%
52%
86%
57%
0%
0%
11/42
5/49
1/79 10/105
60/67
5/5
I to N (% of I)
26%
10%
1%
10%
90%
100%
Number Alive
160
150
130
112
75
67
57
Mean water temperature (oC)
14.4
10.6
12.9
14.7
19.9
22.8
26.2
High
I to N (#)
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Figure Legends
Accepted Article
Figure 1: An experimental mesocosm showing the configuration of refuge habitat.
Figure 2: Mean salinity levels in high (N = 8) and low (N = 8) salt treatment mesocosms
across the study period of 23 weeks. The study began on July 12, 2013 (mid-winter) and
ended December 24, 2013 (mid-summer). Error bars = ± 1 SE.
Figure 3: Mean weekly water temperatures in mesocosms across the study period. The study
began on July 12 (mid-winter) and ended December 24 (mid-summer), 2013. Error bars = ± 1
SD.
Figure 4: Survival (proportion) of frogs released into experimental mesocosms containing
low and high salt concentrations over the course of the study period. (A) All frogs released at
time zero (n = 8 mesocosms and 224 animals per treatment level at time zero); (B) Frogs
released initially without Bd infection only (n = 8 mesocosms and 144 animals per treatment
at time zero); (C) Frogs released initially with Bd infection only (n = 8 mesocosms and 80
animals per treatment at time zero).
Figure 5: Bd prevalence and infection load over time. (A) The proportion of surviving frogs
in low (N = 8) and high (N = 8) salt mesocosms that were infected (tested Bd positive) across
the experimental period; (B) Mean Bd infection loads for all surviving frogs at various time
intervals across the experimental period; (C) Mean Bd infection loads for all surviving frogs
at various time intervals across the experimental period, separated by low and high salt
treatments (N = 8 mesocosms per treatment). Error bars = 95% confidence intervals. GEs =
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genomic equivalents. The study period lasted 23 weeks, from July 12 (mid-winter) to
Accepted Article
December 24 (mid-summer), 2013.
Figures
Figure 1
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Figure 2
High salt
4.5
Salinity (parts per thousand)
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5.0
Low salt
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
5
10
15
Time (weeks)
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20
Figure 3
R² = 0.925
Mean temperature
30
Temperature (degrees celsius)
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35
25
20
15
10
5
0
0
5
10
15
Time (weeks)
This article is protected by copyright. All rights reserved.
20
25
Figure 4
(A)
0.9
Low salt
High salt
0.8
0.7
Surviving
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
Time (weeks)
1
(B)
0.9
Low salt
High salt
0.8
0.7
0.6
Surviving
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1
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
Time (weeks)
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20
1
(C)
Low salt
High salt
0.8
0.7
0.6
Surviving
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0.9
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
Time (weeks)
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20
Figure 5
% of population infected
Infection load (chytrid zoospore GEs)
Infection load (chytrid zoospore GEs)
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100
90
Low salt
80
High salt
70
60
50
40
30
20
10
0
1
5
9
13
17
21
Time (weeks)
600
500
400
300
200
100
0
0
3
6
9
12
Time (weeks)
15
18
21
1400
High salt
1200
Low salt
1000
800
600
400
200
0
0
3
6
9
12
Time (weeks)
15
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18
21